Project Summary Deep brain, high frequency stimulation (HFS) of the Nucleus Accumbens (NAc) is a highly effective treatment for reward-related disorders such as depression. However, the long-lasting mechanisms of HFS and potential non-invasive pharmacological targets of HFS have not been identified. We previously discovered high frequency optogenetic stimulation of dopamine 1 (D1) receptor expressing medium spiny neurons (MSNs) in the NAc mimic deep brain stimulation anti-depressant outcomes. At a synaptic level, HFS of D1-MSNs in the NAc promotes post-synaptic, glutamatergic long-term potentiation on dopamine 2 (D2) receptor expressing MSNs. Higher frequency stimulation parameters are known to promote increased peptide release. Preliminary experiments indicate SP can mimic the D1-MSN HFS-mediated potentiation and the potentiation effect of HFS can be blocked by antagonizing the main receptor for substance P, the neurokinin 1 receptor, suggesting a role for the SP-neurokinin 1 receptor system. However, this effect still remains unclear. Additional preliminary data indicates this effect is likely disynaptic as neurokinin 1 receptors are found in all cholinergic interneurons, but not MSNs, and muscarinic receptor antagonists block the D2-MSN synaptic effects of substance P. We hypothesize NAc D1-MSN HFS causes post-synaptic potentiation of D2-MSN excitatory transmission through SP-induced ChI excitation and subsequent D2-MSN muscarinic 1 receptor signaling These initial experiments describe a multi-synaptic mechanism which will provide multiple pharmacological targets for mimicking NAc HFS. To date, HFS-mediated release of NAc peptides have not been explored at these levels. Utilizing a multi-level approach, I will dissect the mechanism behind frequency dependent, stimulation-mediated substance P release and determine its effects on network activity. I will use optogenetics in combination with whole-cell patch clamp, in vivo microdialysis, and in vivo calcium imaging to determine the frequency-dependent effects of stimulation at each of these levels. My overall aim of this proposal aims to determine peptidergic (substance P) mechanisms underlying therapeutic HFS of the Nucleus Accumbens and provide new pharmacological targets and blood-brain barrier permeable drugs to non-invasively mimic network effects of deep brain HFS.